Preparation method of amniotic membrane hydrogel and amniotic membrane hydrogel

Abstract

The application provides a preparation method of amniotic membrane hydrogel and the amniotic membrane hydrogel, and belongs to the technical field of biological products. The preparation method of the amniotic membrane hydrogel comprises the following steps: (1) amniotic membrane tissue is treated by a decellularization solution containing an antioxidant, and after freeze-drying protection, the amniotic membrane powder is obtained by crushing; (2) the amniotic membrane powder is subjected to enzymolysis by using a neutral protease in a buffer solution with a pH value of 6.5-7.8 to obtain an amniotic membrane active dissolution solution; (3) the amniotic membrane active dissolution solution is mixed with a polysaccharide solution to obtain a mixed solution, and the polysaccharide solution comprises chitosan, beta-glycerophosphate sodium and a nano reinforcing agent; (4) the mixed solution is stored at 2-8 DEG C to form an injectable pre-gel, and the pre-gel is gelled at 25-42 DEG C for 3-10 min. The amniotic membrane hydrogel can better maintain its three-dimensional network structure in actual application by using nano cellulose to enhance the double network structure, and is not prone to damage or deformation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological products, in particular to a preparation method of amniotic membrane hydrogel and the amniotic membrane hydrogel. BACKGROUND

[0002] Amniotic membrane hydrogel is an advanced biomaterial developed based on placental amniotic membrane. It is formed by decellularization and high polymer material compounding to form a three-dimensional network structure with regenerative and repair functions. It usually contains trace elements, various immune factors, active enzymes, body fluid regulating factors, growth factors, and various active polypeptides closely related to immune function and body function. One or several of these substances are usually added to food, medicine, health care products, and cosmetics. Amniotic membrane hydrogel retains collagen, laminin and growth factors (such as EGF and bFGF) in natural amniotic membrane, which significantly promotes cell migration and angiogenesis. The freeze-drying process makes the porosity reach 60-95%, and the water absorption rate reaches 30 times the weight, providing an ideal microenvironment for tissue repair.

[0003] Current amniotic membrane hydrogel is used by compounding with other drugs, such as GelMA-amniotic membrane composite hydrogel: through methacrylation modification to improve mechanical properties, solve the defect of easy degradation of traditional amniotic membrane, and accelerate the healing of full-thickness skin defects. Heat-sensitive vaginal hydrogel: using the heat-sensitive properties of decellularized amniotic membrane to achieve targeted drug delivery, such as reducing systemic side effects when treating premature birth with ritodrine.

[0004] Amniotic membrane hydrogel is mainly used for wound repair, which can improve the healing efficiency by 40% and accelerate the collagen deposition rate by 2.3 times; ophthalmology and gynecology: used for corneal defect repair and premature treatment, with anti-inflammatory and tissue regeneration functions.

[0005] The existing amniotic membrane hydrogel has the problem of insufficient performance, which is prone to damage or deformation in actual application, and cannot well maintain its three-dimensional network structure, affecting its provision of tissue repair microenvironment. Therefore, the preparation method of amniotic membrane hydrogel proposed in the present application is expected to solve the above problems and prepare amniotic membrane hydrogel with better performance. SUMMARY

[0006] The present application is made in view of the above problems, and aims to provide a preparation method of amniotic membrane hydrogel and the amniotic membrane hydrogel.

[0007] Specifically, the present application provides a preparation method of amniotic membrane hydrogel, comprising the following steps:

[0008] (1) The amniotic membrane tissue is treated with a decellularization solution containing an antioxidant, and then crushed after freeze-drying protection to obtain amniotic membrane powder;

[0009] (2) The amniotic membrane powder is enzymatically hydrolyzed with neutral protease in a pH 6.5-7.8 buffer to obtain an amniotic membrane active solution;

[0010] (3) The amniotic membrane active solution is mixed with a polysaccharide solution to obtain a mixed solution, the polysaccharide solution comprising chitosan, β-glycerophosphate sodium and a nano-enhancing agent;

[0011] (4) The mixed solution is stored at 2-8°C to form an injectable pre-gel, which is gelled at 25-42°C for 3-10 min.

[0012] Further, the decellularization solution in step (1) comprises 0.1-0.5‰ EDTA and 1-10 mM ascorbic acid, the treatment temperature is 20-30°C, and / or the treatment time is 1-2 h.

[0013] Further, the freeze-drying protection in step (1) is spraying a solution containing 5-15% trehalose and 0.5-2% human serum albumin, and the freeze-drying condition is main drying at -40 to -50°C for 40-50 h.

[0014] Further, the neutral protease in step (2) is dispersin II or collagenase.

[0015] Further, the temperature for enzymatic hydrolysis in step (2) is 35-38°C, and / or the amount of neutral protease added is 1 / 30-1 / 60 of the mass of the amniotic membrane.

[0016] Further, the composition of the polysaccharide solution in step (3) comprises: chitosan 0.5-1.2% w / v, β-glycerophosphate sodium 10-18% w / v, and nano-enhancing agent 0.05-0.3% w / v.

[0017] Further, the nano-enhancing agent is a biomimetic mineralized nanofiber, which has a core of enzymatically hydrolyzed amniotic membrane collagen nanofiber, and the surface of the core is in-situ mineralized with magnesium silicate nanoparticles.

[0018] Further, the mass ratio of the magnesium silicate nanoparticles to the enzymatically hydrolyzed amniotic membrane collagen nanofiber is 0.5-2:1.

[0019] Further, the enzymatically hydrolyzed amniotic membrane collagen nanofiber has a length of 0.5-5 μm and a diameter of 10-100 nm, and the magnesium silicate nanoparticles have a particle size of 5-50 nm.

[0020] Further, the volume ratio of the amniotic membrane active solution to the polysaccharide solution in step (3) is 2-4:1, and the pH of the mixed solution is adjusted to 6.8-7.2 after mixing.

[0021] The second aspect of the present application provides an amniotic membrane hydrogel prepared by the method for preparing the amniotic membrane hydrogel.

[0022] The present application has the following advantages:

[0023] (1) The preparation process of the present application first treats the amniotic membrane tissue with a decellularization solution containing an antioxidant, which can prevent the active components in the amniotic membrane tissue from being oxidatively damaged during the treatment process, ensuring that the amniotic membrane hydrogel prepared subsequently can retain the original activity of the amniotic membrane tissue to the greatest extent. Then, freeze-drying protection is performed, and trehalose and human serum albumin can provide good protection for the amniotic membrane tissue during freeze-drying, preventing damage to the tissue caused by ice crystal formation and helping to maintain the structure and activity of the amniotic membrane tissue. After freeze-drying protection, the amniotic membrane tissue is crushed into powder, which can increase its specific surface area and facilitate the subsequent enzymatic hydrolysis process. Subsequently, the amniotic membrane powder is subjected to enzymatic hydrolysis with neutral protease in a pH 6.5-7.8 buffer solution, allowing the enzymatic hydrolysis reaction to proceed smoothly. The use of dispersin II or collagenase as neutral protease can decompose the proteins and other components in the amniotic membrane powder, thereby obtaining an amniotic membrane active solution. The amniotic membrane active solution is then mixed with a polysaccharide solution to prepare for the final gelation process. Finally, the mixed solution is stored at 2-8°C to form an injectable pre-gel, which is gelled at 25-42°C for 3-10 min. This temperature-sensitive gelation property gives the amniotic membrane hydrogel a great advantage in practical applications, such as rapid gelation in a specific temperature environment in vivo, better exertion of its tissue repair function, etc.

[0024] (2) The amniotic membrane hydrogel prepared by the present application has significant advantages in performance compared to existing products. By enhancing the double network structure with nanocellulose, the compression modulus of the amniotic membrane hydrogel reaches 8-15 kPa. This improvement in mechanical strength enables the amniotic membrane hydrogel to better maintain its three-dimensional network structure in practical applications, reducing the likelihood of damage or deformation, thereby more effectively providing an ideal microenvironment for tissue repair. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present application clearer, the following embodiments are used to describe and explain the present application. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. Based on the embodiments provided in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0026] It is apparent that the following description is merely some examples or embodiments of the present application, and the present application can also be applied to other similar situations without creative labor for those skilled in the art. In addition, it can be understood that although the efforts made in this development process can be complex and lengthy, some design, manufacture or production changes based on the technology disclosed in the present application are only routine technical means for those skilled in the art related to the disclosure of the present application, and should not be understood as insufficient disclosure of the present application.

[0027] The embodiments of the first aspect of the present application provide a preparation method of amniotic membrane hydrogel, comprising the following steps:

[0028] (1) The amniotic membrane tissue is treated by a decellularization solution containing an antioxidant, and the amniotic membrane powder is obtained by crushing after freeze-drying protection;

[0029] (2) The amniotic membrane powder is enzymolyzed by neutral protease in a buffer solution with pH 6.5-7.8 to obtain an amniotic membrane active dissolution solution;

[0030] (3) The amniotic membrane active dissolution solution is mixed with a polysaccharide solution to obtain a mixed solution, and the polysaccharide solution comprises chitosan, β-glycerophosphate sodium and a nano-enhancing agent;

[0031] (4) The mixed solution is stored at 2-8℃ to form an injectable pre-gel, and the pre-gel is gelled under the environment of 25-42℃ for 3-10min.

[0032] Firstly, the amniotic membrane tissue is treated by a decellularization solution containing an antioxidant, which can prevent the active components in the amniotic membrane tissue from being oxidized and destroyed during the treatment process, ensuring that the amniotic membrane hydrogel prepared subsequently can retain the original activity of the amniotic membrane tissue to the greatest extent. Then, the freeze-drying protection is carried out, and trehalose and human serum albumin can provide good protection for the amniotic membrane tissue during the freeze-drying process, prevent the ice crystals from causing damage to the tissue, and help to maintain the structure and activity of the amniotic membrane tissue. After freeze-drying protection, the amniotic membrane tissue is crushed into powder, which can increase its specific surface area and be beneficial to the subsequent enzymolysis process. Subsequently, the amniotic membrane powder is enzymolyzed by neutral protease in a buffer solution with pH 6.5-7.8, so that the enzymolysis reaction can proceed smoothly. The neutral protease is selected as dispersin II or collagenase, which can decompose the components such as proteins in the amniotic membrane powder, thereby obtaining the amniotic membrane active dissolution solution. Then, the amniotic membrane active dissolution solution is mixed with a polysaccharide solution to prepare for the final gelling process. Finally, the mixed solution is stored at 2-8℃ to form an injectable pre-gel, and the pre-gel is gelled under the environment of 25-42℃ for 3-10min. This temperature-sensitive gelling property makes the amniotic membrane hydrogel have great advantages in actual application, such as rapid gelling in a specific temperature environment in vivo, and better exertion of its tissue repair function.

[0033] In this embodiment, the decellularization solution is prepared by weighing the fresh amniotic membrane after removing the blood clots, immersing it in a 0.1-0.5‰ EDTA solution containing 1-10 mM ascorbic acid (1 L / g of tissue), shaking at 20-30°C for 1-2 h, and rinsing with distilled water for 3 times (reducing matrix loss > 15%). The amniotic membrane is laid flat on a tray, sprayed with a solution containing 5-15% trehalose and 0.5-2% human serum albumin (5 ml / g of tissue), and vacuum freeze-dried: pre-freezing at -45°C for 2 h → main drying at -40°C for 24 h → desorption drying at 25°C for 4 h. The freeze-dried amniotic membrane is cut into pieces, ball-milled under liquid nitrogen (300 rpm, 3×5 min), and sieved through a 300-mesh sieve to obtain amniotic membrane powder (particle size < 50 μm), which is sealed and stored away from light. This step effectively removes the cellular components in the amniotic membrane tissue while maximizing the retention of active ingredients and structural integrity of the amniotic membrane tissue. The use of antioxidants prevents the oxidation of active ingredients, and the freeze-drying protection measures avoid ice crystal damage, and the crushing process increases the specific surface area, creating favorable conditions for subsequent enzymatic hydrolysis.

[0034] In this embodiment, step (2) involves adding 1 g of amniotic membrane powder to 20 ml of 0.1 M PBS buffer with a pH of 7.4, pre-swelling at 35-38°C for 30-40 min, adding Dispase II or collagenase, preferably Dispase II, and enzymatically hydrolyzing at 35-38°C for 24 h. The amount of Dispase II added is 1 / 30-1 / 60 of the mass of the amniotic membrane. This step uses neutral protease to enzymatically hydrolyze the amniotic membrane powder in a suitable pH buffer and temperature condition, allowing the proteins and other components in the amniotic membrane powder to be effectively broken down. Taking Dispase II as an example, it can specifically act on the proteins in the amniotic membrane, cutting them into small molecular fragments and forming an amniotic membrane active dissolution solution. During the enzymatic hydrolysis process, the temperature is controlled at 35-38°C, preferably 37°C, because this temperature range is close to the human body temperature, allowing the protease to maintain high activity and promoting the smooth progress of the enzymatic hydrolysis reaction. The enzymatic hydrolysis time is set to 24 h to ensure that the amniotic membrane powder is fully broken down, thereby obtaining a sufficient amount of amniotic membrane active dissolution solution with stable quality. Meanwhile, the amount of Dispase II added is 1 / 30-1 / 60 of the mass of the amniotic membrane, which has been verified through a large number of experiments to ensure the enzymatic hydrolysis effect while avoiding excessive decomposition and cost increases due to excessive addition of protease. After the enzymatic hydrolysis is completed, impurities that have not been completely broken down can be removed by filtration and other methods, further improving the purity and quality of the amniotic membrane active dissolution solution and laying a good foundation for subsequent mixing with the polysaccharide solution and final gel formation.

[0035] In the present embodiment, the composition of the polysaccharide solution in step (3) includes: chitosan 0.5-1.2% w / v, β-glycerophosphate sodium 10-18% w / v, and nano-enhancing agent 0.05-0.3% w / v. Preferably, the chitosan is deacetylated chitosan, the deacetylated chitosan is 0.8% w / v, the β-glycerophosphate sodium is 15% w / v, and the nano-enhancing agent is 0.1% w / v.

[0036] In the present embodiment, the nano-enhancing agent is a biomimetic mineralized nanofiber, which has a core of enzymatic amniotic membrane collagen nanofiber and a surface in-situ mineralized with magnesium silicate nanoparticles; the mass ratio of the magnesium silicate nanoparticles to the enzymatic amniotic membrane collagen nanofiber is 0.5-2:1. The enzymatic amniotic membrane collagen nanofiber has a length of 0.5-5 μm and a diameter of 10-100 nm; the magnesium silicate nanoparticles have a particle size of 5-50 nm.

[0037] The preparation method of the nano-enhancing agent is S1: after the amniotic membrane active solution prepared in step (2) is dialyzed and purified, enzymatic amniotic membrane collagen nanofiber having a length of 1-2 μm and a diameter of 20-50 nm is prepared by electrospinning-high-pressure cutting technology, dispersed in deionized water to have a concentration of 1% (w / v), and an enzymatic amniotic membrane collagen nanofiber suspension is prepared; S2: a 0.1M MgCl2 solution is prepared, the enzymatic amniotic membrane collagen nanofiber suspension is mixed with the MgCl2 solution, Mg 2+ : collagen amino molar ratio = 2:1; 4℃ stirring for 12h, so that Mg 2+ is combined with the negative groups on the collagen fiber; S3: 0.06M Na2SiO3 solution (SiO3 2- : Mg 2+ molar ratio = 1:1) is slowly added dropwise, pH is maintained at 9.0 by dilute NaOH, and 40℃ water bath reaction is performed for 24h; centrifugal washing and freeze-drying are performed to obtain the nano-enhancing agent.

[0038] The present application uses organic nanofiber (collagen) as a template, in-situ mineralizes inorganic nanoparticles (magnesium silicate) on the surface of the organic nanofiber, and forms an organic-inorganic hybrid nano-enhancing agent. Not only mechanical enhancement is provided, but also multiple biological activities such as antibacterial, pro-angiogenic, self-healing, etc. are endowed to the hydrogel. The degradation products (Mg 2+ , SiO3 2- ) of the magnesium silicate have clear biological activities: Mg 2+ promotes angiogenesis, and SiO3 2-Promote collagen deposition and osteogenic differentiation. On the one hand, the nano-enhancer constructs an organic-inorganic hybrid structure by in-situ mineralizing magnesium silicate nanoparticles on the surface of enzymolyzed amniotic membrane collagen nanofibers, which greatly enhances the mechanical properties of amniotic membrane hydrogel. Its compressive modulus reaches 8-15 kPa, enabling the amniotic membrane hydrogel to better maintain a three-dimensional network structure in practical applications, and not easily broken or deformed, providing a stable microenvironment for tissue repair. On the other hand, the nano-enhancer endows the amniotic membrane hydrogel with rich biological activity. The degradation products of magnesium silicate, Mg 2+ and SiO3 2- , play an important role. Mg 2+ has the function of promoting angiogenesis, and in the process of tissue repair, it can stimulate the proliferation and migration of vascular endothelial cells, promote the formation of new blood vessels, provide sufficient oxygen and nutrients for damaged tissues, and accelerate the repair and regeneration of tissues. SiO3 2- can promote collagen deposition and osteogenic differentiation. In bone tissue repair, it can induce mesenchymal stem cells to differentiate into osteoblasts, promote the synthesis and mineralization of bone matrix, and help repair and heal bone defects. At the same time, SiO3 2- can also promote the synthesis and secretion of collagen, enhance the toughness and strength of the tissue. In addition, the nano-enhancer also makes the amniotic membrane hydrogel have the characteristics of antibacterial and self-healing. In the process of tissue repair, the antibacterial property can effectively prevent bacterial infection and reduce inflammatory response, creating a good environment for tissue repair. The self-healing function can make the amniotic membrane hydrogel automatically repair the damaged part after being slightly damaged, maintain the integrity of its structure and function, prolong its service life and improve its use effect.

[0039] The preparation method of the polysaccharide solution is: dissolving chitosan in 0.1M acetic acid buffer, adjusting the pH to 5.0, stirring overnight, adding β-glycerophosphate sodium to dissolve, then heating the nano-enhancer, ultrasonic dispersion (100W, 30min), adjusting the pH to 7.0±0.2 after mixing, and storing at 4℃.

[0040] The amniotic active dissolution solution and the polysaccharide solution are mixed at a volume ratio of 2-4:1, and 0.05% volume of recombinant human hyaluronidase inhibitor is added. Dropwise mixing under ice bath conditions, and adjusting the pH to 6.8-7.2 after mixing. This step mixes the amniotic active dissolution solution and the polysaccharide solution to form a uniform mixture, and the added recombinant human hyaluronidase inhibitor can effectively inhibit the activity of hyaluronidase, preventing the components in the amniotic active dissolution solution and the polysaccharide solution from being excessively decomposed, thereby ensuring the stability and activity of the mixture. Dropwise mixing under ice bath conditions and standing at 4℃ for 1h helps the components in the mixture to interact fully, making the mixture more uniform, laying a foundation for the formation of an injectable pre-gel with excellent performance and the final amniotic membrane hydrogel.

[0041] When the mixed solution is stored at 2-8℃, due to the presence of chitosan, β-glycerophosphate sodium and nano-enhancing agent in the polysaccharide solution, an injectable pre-gel will gradually form. Chitosan has good fluidity at low temperature, while β-glycerophosphate sodium as a crosslinking agent slowly reacts with chitosan at low temperature, causing the viscosity of the mixed solution to gradually increase, forming a pre-gel state with certain fluidity but also maintaining the shape. The addition of nano-enhancing agent further enhances the structural stability and mechanical properties of the pre-gel, allowing it to better maintain its shape and not easily deform during subsequent injection.

[0042] When the injectable pre-gel is placed in an environment of 25-42℃, it can quickly gel within 3-10min. This is because the crosslinking reaction between chitosan and β-glycerophosphate sodium accelerates in this temperature range, forming a more compact three-dimensional network structure. The nano-enhancing agent is uniformly distributed in this network structure, further enhancing the mechanical properties of the amniotic membrane hydrogel. This rapid gelation property allows the amniotic membrane hydrogel to quickly adapt to the in vivo environment in practical applications, such as in the repair of corneal defects in ophthalmology or the treatment of premature birth in gynecology, providing stable support and a good microenvironment for tissue repair.

[0043] The second aspect of the present application provides an amniotic membrane hydrogel prepared by the preparation method of the amniotic membrane hydrogel.

[0044] This amniotic membrane hydrogel has good biocompatibility and biological activity, and has a wide application prospect in the field of biomedicine.

[0045] Examples

[0046] The following examples more specifically describe the present disclosure, which are merely illustrative and not intended to limit the scope of the present disclosure, as various modifications and variations will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight. Unless otherwise stated, all reagents used in the examples are available through conventional commercial channels or synthesized according to conventional methods and used as received without further purification. Unless otherwise stated, the instruments used in the examples are available through conventional commercial channels.

[0047] Example 1

[0048] A preparation method of an amniotic membrane hydrogel, comprising the following steps:

[0049] (1) Take 100g of fresh amnion, scrape off the blood clots and weigh it, soak it in 0.2‰ EDTA solution containing 5mM ascorbic acid (1L / g tissue), shake at 25℃ for 1.5h, rinse 3 times with distilled water, lay the amnion flat on a tray, spray it with a solution containing 10% trehalose and 1.2% human serum albumin (5ml / g tissue), freeze dry at -45℃ for 24h, and ball mill through a 300-mesh sieve to obtain amnion powder;

[0050] (2) 1g of amniotic membrane powder was added to 20ml of 0.1M PBS buffer, pH 7.4, pre-swelled at 37℃ for 35min, 20mg of dispersible protease II was added, enzymatically digested at 37℃ for 24h, inactivated at 70℃, and centrifuged to obtain amniotic membrane active solution;

[0051] (3) Preparation of polysaccharide solution: 0.8% chitosan, 15% sodium β-glycerophosphate, 0.1% nano-enhancing agent; mix amniotic membrane active dissolving solution and polysaccharide solution at a volume ratio of 3:1 to obtain a mixed solution, adjust pH to 7.0, and store at 4℃;

[0052] (4) The mixture is stored at 4°C to form an injectable pregel, and then gelled at 37°C for 3 min 20 s.

[0053] Example 2

[0054] This embodiment is basically the same as that of embodiment 1, except that a 0.4‰ EDTA solution containing 8mM ascorbic acid is used in step (1).

[0055] Example 3

[0056] This embodiment is basically the same as that of embodiment 1, except that the polysaccharide solution in step (3) contains 1% chitosan, 12% sodium β-glycerophosphate, and 0.15% nano-reinforcing agent.

[0057] Example 4

[0058] This embodiment is basically the same as that of embodiment 1, except that in step (3), the amniotic membrane active dissolving solution and the polysaccharide solution are mixed at a volume ratio of 4:1.

[0059] Example 5

[0060] This embodiment is basically the same as that of embodiment 1, except that the concentration of sodium β-glycerophosphate in step (3) is 18%.

[0061] Comparative Example 1

[0062] This comparative example is basically the same as Example 1, except that in step (2), 1 g of amniotic membrane powder is added to 10 ml of 0.01 M HCl (pH 2.0) and 100 mg of pepsin, and digested at 37°C for 72 h; 1 ml of 0.1 M NaOH is added for neutralization, and then mixed with 15% Pluronic F127 solution at a ratio of 1:1 to prepare a hydrogel.

[0063] Comparative Example 2

[0064] This comparative example is basically the same as Example 1, except that no nano-enhancing agent is added to the temperature-sensitive solution.

[0065] Comparative Example 3

[0066] This comparative example is basically the same as Example 1, except that pepsin is used instead of Dispase II (same enzyme amount).

[0067] Comparative Example 4

[0068] This comparative example is basically the same as Example 1, except that the concentration of β-GP is reduced to 8%.

[0069] Experimental Cases

[0070] The amniotic membrane hydrogels in Examples 1-5 and Comparative Examples 1-4 are gelled in a 37°C water bath, and the gelling time is recorded; a 26G needle is used for injection force testing; and a universal testing machine is used for compression modulus testing, and the results are shown in Table 1.

[0071]

[0072] As can be seen from Table 1, the amniotic membrane hydrogels prepared in Examples 1-5 are overall superior to Comparative Examples 1-4 in terms of gelling time, injection force, and compression modulus. In terms of gelling time, the gelling time of the examples is generally shorter, mostly around 3-4 minutes, while the gelling time of the comparative examples is significantly longer. This shows that the preparation method used in the present application, especially the formulation of the polysaccharide solution and the mixing method with the amniotic membrane active dissolution solution, can make the amniotic membrane hydrogel gel in a shorter time, and can play a faster role in actual application.

[0073] In terms of injection force, the injection force of the examples is relatively small, indicating that it has good fluidity and injectability. This is due to the good fluidity of chitosan in the polysaccharide solution at low temperature and the enhancement of the nano-enhancing agent on the structure stability and mechanical properties of the pre-gel. The injection force of the comparative examples is larger, which may require more force during injection, increasing the difficulty of operation and possibly causing more damage to the tissue.

[0074] The compression modulus reflects the mechanical properties of the amniotic membrane hydrogel. The compression modulus of the examples is high, indicating that it can provide more stable support for tissue repair. The addition of nano-enhancing agents further enhances the mechanical properties of the amniotic membrane hydrogel, enabling it to better maintain its shape in the body and adapt to the needs of tissue repair. The compression modulus of the comparative examples is relatively low, which may not provide sufficient support for tissue repair, affecting the repair effect.

[0075] In summary, the preparation method of the amniotic membrane hydrogel provided by the present application has significant advantages. By reasonably selecting the decellularization solution, freeze-drying protectant, enzymatic conditions, polysaccharide solution formula, and mixing method, etc., the prepared amniotic membrane hydrogel performs well in terms of gelation time, injection force, and compression modulus, etc., has good biocompatibility and biological activity, and has a wide application prospect in the biomedical field. It is expected to provide more effective treatment methods for ophthalmic corneal defect repair, gynecological premature treatment, etc.

[0076] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and embodiments having substantially the same configuration and playing the same role and effect as the technical idea within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the main idea of the present application, various modifications of the embodiments that can be thought of by those skilled in the art, and other ways constructed by combining part of the constituent elements of the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing an amniotic membrane hydrogel, characterized by, The method comprises the following steps: (1) The amniotic membrane tissue is treated with a decellularization solution containing an antioxidant, and the amniotic membrane powder is obtained by freeze-drying and crushing after protection; (2) The amniotic membrane powder is enzymatically hydrolyzed with neutral protease in a buffer solution with a pH of 6.5-7.8 to obtain an amniotic membrane active solution; The neutral protease is dispersin II or collagenase; (3) The amniotic membrane active solution is mixed with a polysaccharide solution to obtain a mixed solution, wherein the polysaccharide solution contains 0.5-1.2% w / v chitosan, 10-18% w / v β-glycerophosphate sodium, and 0.05-0.3% w / v nano-enhancing agent; The nano-enhancing agent is a biomimetic mineralized nanofiber, which has an enzymatically hydrolyzed amniotic membrane collagen nanofiber as a core, and magnesium silicate nanoparticles are in-situ mineralized on the surface of the core; (4) The mixed solution is stored at 2-8°C to form an injectable pre-gel, and the pre-gel is gelled at 25-42°C for 3-10 min.

2. The method of claim 1, wherein the amniotic membrane hydrogel is prepared by the steps of: In step (1), the decellularization solution contains 0.1-0.5‰ EDTA and 1-10 mM ascorbic acid, the treatment temperature is 20-30°C, and / or the treatment time is 1-2 h.

3. The method of claim 1, wherein the amniotic membrane hydrogel is prepared by the steps of: In step (1), the freeze-drying protection is spraying a solution containing 5-15% trehalose and 0.5-2% human serum albumin, and the freeze-drying conditions are -40 to -50°C for 40-50 h.

4. The method of claim 1, wherein the amniotic membrane hydrogel is prepared by the steps of: In step (2), the enzymatic hydrolysis temperature is 35-38°C, and / or the amount of neutral protease added is 1 / 30-1 / 60 of the mass of the amniotic membrane.

5. The method of claim 1, wherein the amniotic membrane hydrogel is prepared by the steps of: The mass ratio of the magnesium silicate nanoparticles to the enzymatically hydrolyzed amniotic membrane collagen nanofiber is 0.5-2:

1.

6. The method of claim 1, wherein the amniotic membrane hydrogel is prepared by the steps of: The enzymatically hydrolyzed amniotic membrane collagen nanofiber has a length of 0.5-5 μm and a diameter of 10-100 nm, and the magnesium silicate nanoparticles have a particle size of 5-50 nm.

7. The method of claim 1, wherein the amniotic membrane hydrogel is prepared by the steps of: In step (3), the volume ratio of the amniotic membrane active solution to the polysaccharide solution is 2-4:1, and the pH is adjusted to 6.8-7.2 after mixing.

8. An amniotic membrane hydrogel, characterized in that, The amniotic membrane hydrogel is prepared by the method of any one of claims 1-7.

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